A recent review by Widdel (1986) in Anaerobic Bacteria in Habitats Other Than Man, pp 157-184, Eds. Barnes and Mead, Blackwell Scientific Publications, includes as sulfate-reducing bacteria the genera: Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfobulbus, Desulfococcus, Desulfonema, Desulfosarcina and Thermodesulfobacterium. Significant inter- and even intra-generic differences exist in terms of morphologies, ecological niches, and metabolic capabilities. However, all respiratory sulfate-reducing bacteria are strict anaerobes which are poisoned by oxygen. The preponderance of isolates are eubacterial and classified as Desulfovibrio although extremely thermophilic archaebacterial sulfate-reducers have been isolated from undersea volcanoes (Stetter et al., 1987 Science 236:822-825). Thus, the term sulfate-reducer encompasses a broad spectrum of organisms across both eubacterial and archaebacterial branches of bacterial phylogeny.
The metabolic capabilities of the genus Desulfovibrio reflect the range of available niches. Utilization of sulfate as an electron sink and respiratory substrate is the characteristic and predominant mode of energy generation. However, nitrate, elemental sulfur, fumarate and other sulfur oxy-acids, when present, can also serve in a similar capacity for a few species. These bacteria are specialists adapted to use the metabolic end-products of primary degradative bacteria for electron and carbon sources as described by Odom et al., 1981 in Trends in the Biology of Fermentations for Fuels and Chemicals, Eds. Hollaender, Rabson, Rogers, Pietro, Valentine, and Wolfe, Plenum Publishing and Odom et al., 1984 Ann. Rev. Microbiol. 38:551-592. Organic acids such as lactate, formate, and pyruvate, alcohols such as ethanol, and molecular hydrogen are the preferred electron sources for sulfate reduction. Acetate, which is the metabolic end-product of the Desulfovibrio, can be utilized by Desulfobacter as an electron donor with carbon dioxide as the sole end product (Stetter et al., 1987 Science 236:822-825). Thus, the sulfate-reducers can effect total mineralization of organic matter from the level of alcohols and acids to carbon dioxide.
The sulfate-reducers play a special role in methane formation as it occurs in sewage treatment and freshwater bogs. In these situations, where sulfate concentrations are very low, the sulfate-reducer enters into a symbiotic relationship with methane-producing bacteria (methanogen) wherein the sulfate-reducers actually produce hydrogen from organic acids and alcohols only if the hydrogen is continuously consumed by the methanogen. This important process, termed interspecies hydrogen transfer, is a vital link in the food chain from complex polymers to methane (Odom et al., 1981 in Trends in the Biology of Fermentations for Fuels and Chemicals, Eds. Hollaender, Rabson, Rogers, Pietro, Valentine, and Wolfe, Plenum Publishing). The most intensively studied aspect has been hydrogen metabolism due to its inherent relationship with methane (fuel) generation from biomass. Similarly, the implication of microbial hydrogen uptake in the phenomenon of anaerobic corrosion of steel according to the theory of cathodic depolarization has stimulated research into the the hydrogen metabolism of sulfate-reducers as well as other microbial types (Von Wolzogen Kuhr et al., 1934 Water 18:147-165, Odom et al., 1984 Ann. Rev. Microbiol. 38:551-592, Pankhania et al., 1986 J. Gen. Microbiol. 132:3357-3365, and Ringas et al., 1987 Corrosion Engineering 44 #6:386-396).
There are currently approximately 130 different industrial biocide products registered with the U.S. Environmental Protection Agency which are produced by over a 100 different companies in the U.S. alone. The leading biocides are halogenated compounds, which make up 35% of total sales, while quarternary detergents and phenolics represent 22% of total sales. Organometallic compounds, inorganic compounds, aldehydes, anilides, and organosulfur compounds make up the rest of the total. None of the known commercial chemicals are specific for sulfate-reducing bacteria, but many have demonstrated effectiveness against sulfate-reducers. Below are discussed some of the more commonly used inhibitors against sulfate-reducing bacteria.
Organo-sulfur or sulfur-nitrogen compounds contain some of the more effective industrial biocides against sulfate-reducers. The isothiazoline containing compounds (produced by Rohm & Haas, Inc. under the name "Kathon.RTM.") are effective against, but not specific for, sulfate-reducers at 12-ppm (Oil and Gas Journal Mar. 8, 1982, p 253). The thiocyanate containing compounds are effective in the range of 5-30 ppm (i.e. "Cytox.RTM."). 2,2-Dibromo-3-nitrilopropionamide (DBNPA) is produced by Nalco, Inc. and is claimed to be particularly effective against sulfate-reducing bacteria at 3-12 ppm. This compound is also a good surfactant and corrosion inhibitor. Recently, Nalco, Inc. has introduced an oilfield biocide claimed to be effective against sulfate-reducers. The product is essentially metronidazole, which is a pharmaceutical originally used to treat Trichosomal infections. The compound is not specific for sulfate-reducers but is claimed to be generally effective against anaerobic bacteria.
Acids and aldehydes are also effective inhibitors of sulfate-reducers. Glutaraldehyde is widely used in water flooding situations at concentrations in the range of 100 to 2000 ppm. The sole U.S. producer is Union Carbide, Inc. with its own registered "Ucarcide.RTM." formulation. Ucarcide.RTM. is claimed to protect oil fields from aerobic and anaerobic microorganisms including sulfate-reducing bacteria in water flooding situations, injection water, drilling and packer fluid. Formaldehyde, which is substantially cheaper, is also used in similar concentrations. These two compounds together comprise the major biocides used in the oil field. Acrolein, an extremely toxic compound, is also effective against sulfate-reducers and is reccommended for use at concentrations in the range of 1 to 15 ppm.
Quarternary amines are a diverse group of compounds containing a quarternary nitrogen atom with long chain alkyl or aromatic substituents. In the oil field these compounds may act as both corrosion inhibitors and bacteriostatic agents. The compounds are used in this application in the 5-100 ppm range. At low concentrations these compounds may be bacteriostatic rather than bacteriocidal. The quarternary amines are generally less hazardous than many oil field biocides, but they must often be used in conjunction with more toxic biocides to enhance their effectiveness.
Halogenated compounds, such as chlorhexidine (a biguanide), (Hibitane.RTM. from ICI, Inc.) is a widely used, commercial, antimicrobial compound, and is known to be effective against a number of sulfate-reducing bacteria at concentrations of 1-10 ppm (Davies et al., 1954 Brit. J. Pharmacol. 9:192). This compound or its derivatives are generally effective against most bacterial types and are, therefore, nonspecific. Biguanides such as chlorhexidine appear to function by disruption of the cell membrane which causes release of cytoplasmic contents. There is no available data on any industrial use of this chemical to treat sulfate-reducing bacteria.
Inorganic compounds, such as liquid chlorine, hypochlorites, chlorine dioxide and chloroisocyanurates are strong oxidizing agents, are often found as the active constituents in bleach, and have shown effectiveness in oil field situations against sulfate-reducing bacteria.
Classical inhibitors of sulfate reduction such as molybdate, selenate, and fluorophosphate anions are analogues of sulfate and have been shown to interfere with the primary enzymatic step in the activation of sulfate, i.e., the adenosine 5'-triphosphate (ATP) sulfurylase reaction. Here an unstable phospho-analogue anhydride is formed in place of the phospho-sulfate bond. The consequence of this is that the bacterium eventually depletes its energy reserve of ATP via reaction with these analogues and death ensues (Taylor et al., 1979 Current Microbiol. 3:101-103 and Wilson et al., 1958 J. Biol. Chem. 233:975-981). Sulfate analogues have been used to inhibit sulfate-reduction at concentrations from 5-20 mM (i.e., 1000-4000 ppm molybdate). These levels are impractical from an applications standpoint but the compounds have found use as research tools (Postgate, 1952 J. Gen. Microbiol. 6:128-142 and Saleh et al., 1964 J. Appl. Bact. 27#2:281-293). Furthermore, the sulfate analogues inhibit sulfate assimilation as it occurs in all bacteria and plants, as well as sulfate respiration, and, thus, are not truly specific for sulfate-reducing bacteria.
Antibacterial activity associated with anthraquinones was first discovered in plant extracts of the genus Cassia. (Patel et al., 1957 Indian J. Pharmacol. 19:70-73). Subsequent investigations revealed that the active component of leaf extracts of Cassia sp. was Rhein or 4,5-dihydroxyanthraquinone-2-carboxylic acid (Anchel, 1949 J. Biol. Chem. 177: 169-177) . Subsequently, it has become apparent that many anthraquinones have antibacterial properties, however, it is equally clear that these compounds do not inhibit all bacterial types. In one study by F. Kavanaugh it would appear that gram positive organisms such as Bacillus sp. or Staphylococcus are sensitive to anthraquinones while gram negative species such as E. coli or Pseudomonas sp. are rather insensitive (Kavanaugh 1947 J. Bacteriol. 54:761-767). However, even among the gram positive bacteria, antibacterial effects are sporadic and unpredictable. For example, another study showed that 1,4,6,8-tetrahydroxyanthraquinone inhibited 4 species of Bacillus, one strain of Nocardia, one strain (out of four tested) of Streptomyces and one of the Gram negative Proteus. The compound did not affect any species of E. coli, Pseudomonas, Salmonella or Sarcina (Anke et al., 1980 Arch. Microbiol. 126:223-230 and Anke et al., 1980 Arch Microbiol., 126:231-236). A study by Bakola-Christianopoulou et al. 1986 Eur. J. Med. Chem.-Chim. Ther. 21#5:385-390, where the metal chelates of the anthraquinones were studied, showed that 1,8-dihydroxyanthraquinone was inactive against B. aureus, B. subtilis, B. stearothermophilus and S. aureus. In the same study 1,2-dihydroxyanthraquinone and 1-amino-4-hydroxyanthraquinone were either inactive against these strains or required concentrations in excess of 100 to 1000 ppm for inhibition. These workers also concluded that the metal chelates were more active than the free uncomplexed compounds and that the compounds showed the most activity against the gram positive Bacillus sp. These results are typical of the published studies on the antibacterial activity of anthraquinones.
Swiss Patent No. 614,466 of Mycogel Laboratories Inc., Brooklyn, N.Y., entitled Agent for Inhibiting the Growth of Bacteria in Culture Media and Use of the Agent describes the use of compounds derived from paraquinone or their hemiquinone or glycoside derivatives as agents for use in culture media for cultivating fungi and yeasts. The rationale disclosed was that these compounds inhibit bacterial growth but not the growth of eukaryotic cells such as molds and yeasts. Preferred anthraquinone derivatives claimed include those substituted with methyl, hydroxymethyl, carboxyl, aldehyde and carboxyethyl groups. Haran et al., 1981 Isr. J. Med. Sci. 17#6:485-496, demonstrated that certain diaminoanthraquinone derivatives exhibited toxicity against gram positive cocci and that gram negative bacteria were rather insensitive.
The mode of action of anthraquinones on bacterial metabolism is not clear and may be multitudinous. It is clear that the inhibitory effect is only observed with bacteria and not with plant, fungal or mammalian tissue, hence, the compounds are relatively non toxic to higher life forms. It is known that many anthraquinones interfere with bacterial DNA metabolism, presumably at the site of DNA directed RNA polymerase (Anke et al., 1980 Arch Microbiol., 126:231-236). Anthraquinone-containing compounds have also been shown to inhibit mitochondrial ADP transport (Boos et al., 1981 FEBS Lett. 127:40-44). It is also known that reduced anthraquinones may react chemically with oxygen to produce the highly toxic superoxide radical and this is generally very toxic to bacteria (Shcherbanovskii et al., 1975 Rastit. Resur. 11#3:445-454).
The consensus from the existing literature is that anthraquinones are not generally antimicrobial. The organisms that are sensitive to anthraquinones have been Gram positive bacteria, in particular Bacillus sp. The anthraquinones do exhibit an array of unrelated and unpredicted biological effects as briefly listed above. The inhibition of sulfide production by anthraquinones is unreported in the literature and appears to be another example of an unpredicted and unrelated biological effect, particularly considering that the sulfate-reducers are Gram negative organisms.
It is clear that sulfide pollution is a growing industrial and environmental concern for which there exists no truly effective or adequate treatment that is environmentally sound. The chemical treatments that are available have a number of shortcomings. Many of these chemicals are highly reactive with short effective lives as antimicrobials and therefore high concentrations are required. Secondly, due to their inherent toxicity, these compounds may pose a health hazard to the personnel using them. Thus a need exists for better means of controlling sulfide pollution.
One key and important difference between existing chemicals and the compounds of the instant invention is the relatively unreactive nature of a number of anthraquinones as a group. In fact, 1,8-dihydroxyanthraquinone (one of our most potent compounds) has been sold commercially as a laxative (see Physicians' Desk Reference, page 574 for Modane.RTM., and page 575 for Modane Plus.RTM.). Many anthraquinones, including derivatives of 1,8-dihydroxyanthraquinone, are naturally-occurring in a number of plants such as rhubarb (Rheum officinale). Use of plant extracts include senna in Senakot.RTM. Tablet and Senokot-S.RTM. (see Physician's Desk Reference, pages 1596-1597), and casenthranol in Dialose Plus.RTM. (see Physicians' Desk Reference, page 1979).
The specific inhibitory activity of the compounds of the present invention and their lack of toxicity to many other organisms opens up entirely new possibilities for use in various waste treatment situations where conventional biocides cannot be used. For example, the odor associated with sulfide pollution in sewage treatment is both a health hazard and an aesthetic problem for many communities. More significantly, from the economic standpoint, concrete conduits are damaged from the aerobic oxidation of sulfide to sulfuric acid. This is a particular problem for cities such as Miami or Los Angeles where the distances involved in sewage transit at ambient temperatures mean long residence times, high metabolic activity and tremendous destruction of concrete structures. The current "state of the art" method to treat this problem is to precipitate the sulfide from solution using massive amounts of ferrous chloride. This alleviates the odor problem but does not remove the sulfide as a substrate for acid producing bacteria (Jameel 1988 Journal WPCF 61#2:230 and Dezham et al., Journal WPCF 60#4:514). The instant invention is particularly advantageous for this kind of application.